The detection, identification, and quantification of nucleic acids are fundamental to many diagnostic tests for infection, disease, contaminants, and/or allergens. In these tests, the diagnosis and sometimes prognosis of a pathological condition is based on the presence of minute quantities of nucleic acids from a microorganism or a cell. Identification of multiple pathogens and their phenotypes as well as one or more specific gene mutations and quantification of nucleic acids, particularly from microorganisms, are also important steps in establishing the cause of an infection [Cornett et al., Angew Chem Int Ed Engl, 2012, 51, 9075-9077; Murray & Salomon, Proc. Natl. Acad. Sci USA, 1998, 95, 13881-13886. Malanoski et al. Nucleic acids res., 2006, 34, 5300-5311]. Similarly, the identification and quantification of single nucleotide polymorphisms (SNPs) in genes like BRCA, p53, and KRAS provides information about cancer and other diseases.
While scientific research contributes to the breadth of diagnostic tests, speed and convenience play an increasingly prominent role in the testing methods. Multiplex assays, in which multiple analytes are measured in a single run/cycle of the assay, and point of care (POC) testing, in which tests are performed at or near the site of patient care, are expected to deliver fast and affirmative results [Gervais et al., Adv Mater, 2011, 23, H151-H176]. For nucleic acid analysis, techniques such as DNA melting analysis using high resolution instrumentation and specialized fluorescent DNA-binding dyes are used to determine the presence and identity of different nucleic acids in the same solution. Another technique based on qPCR powerfully combines sensitive nucleic acid detection in real-time with multiplexing capacity [Espy et al., Clin Microbiol Rev, 2006, 19, 165-256]. However, these techniques require the use of multiple fluorescent dyes to monitor the amplification of target nucleic acids in real-time [Lodeiro et al., Chem Soc Rev, 2010, 39, 2948-2976]. Because resonance energy transfer occurs between the dyes, the sensitivity of these reactions is hampered, and most qPCR assays can simultaneously detect only 2-3 targets [Wittwer et al., Methods, 2001, 25, 430-442]. Conventional PCR performed without dyes could support many more targets, as evidenced by experiments in which the characteristic melting temperature (Tm) of PCR amplicons were used as a secondary label in qPCR, leading to identification of 50 different DNA sequences in one sample [Mackay et al. Nucleic acids res., 2002, 30, 1292-1305; Liao et al., Nucleic acids res., 2013, 41, e76]. Moreover, although multiplex DNA detection based on the melting temperature (Tm) analysis has been applied many times in PCR assays, the melting analysis is always applied post PCR, and does not allow for monitoring of specific targets during the PCR reaction [Li et al. Nucleic acids res., 2007, 35, e84]. As a result, the melting analysis can be used to detect the presence of DNA targets, but cannot be used to quantitate DNA. These limitations make the technology better-suited as a screening tool for the presence of mutations, but there remains a need in the art for techniques which enable rapid, sensitive, and accurate identification and quantification of nucleic acid targets.
To monitor DNA amplification during PCR reactions, DNA melting can be followed in real-time using surface plasmon resonance (SPR) devices. An SPR imaging device was used to discriminate between SNPs in short target sequences, [Fiche et al., Analytical Chemistry, 2008, 80, 1049-1057] while hybridization of Au NPs to the surface of the SPR devices improved resolution so that SNPs were discriminated in longer targets (PCR amplicons) [Knez et al., Small, 2012, 8, 868-872]. Herein a PCR reaction is performed with one primer pair to detect different mutations within the same target nucleic acid. After completion of the amplification reaction the presence of different mutated forms is detected in a separate sensor device by determining the melting temperature of the different mutants.
For the latter studies, a fibre optic SPR (FO-SPR) device was used as a “dip probe” to test different solutions. The FO-SPR device was used primarily for monitoring solid phase PCR amplification reactions, although efficiency was limited [Pollet et al. Small, 2011. 7, 1003-1006] In this method the solid phase PCR reaction is performed using one primer which is attached to the sensor and one primer which is attached to a gold particle. The probes on the surface are extended during PCR and determination of the melting point is thus dictated by the behaviour of the entire amplicon and not just by primer template hybridisation.
Multiplex PCR reactions would only be possible by using multiple fibres (i.e., an individually functionalized fibre for each amplification reaction).
Delport et al. (2012) Nanotechnology 23, 065503, measures the melting point of a double stranded DNA wherein one strand is attached to the surface of a fibre optic sensor and the second strand is attached to a silica nanoparticle.